ATP1B3负调控巨噬细胞NLRP3炎症小体在脓毒症中的作用机制研究

Excessive NLRP3 inflammasome activation induced over-production of inflammatory cytokine and pyroptosis of immune cells, which plays pivotal roles in the course of disease. It is important to find drugs which negative-regulated NLRP3 inflammasome and elucidate corresponding mechanism. We had reported that chloroquine dampened NLRP3 inflammasome in vitro and in vivo, and improve survival rate in sepsis models, while the mechanism is unclear. Preliminary experiment found that potassium efflux decrease dramatically by chloroquine. Results from Affymetrix GeneChip mouse Transcriptome Assay 2.0 prompt Potassium ion transporters ATP1B3 up-regulated by chloroquine. Combined with the literature, we proposed that up-regulated ATP1B3 play a negative regulation role in NLRP3 inflammasome activation in macrophage, decrease cytokines release and pyroptosis, and then protected sepsis model. In order to verify the assumption, the study intend to conduct a research that ATP1B3 acted on NLRP3 inflammasome through signal 1 pathway and signal 2 pathway in cell models and sepsis animal models, then protectived of sepsis animal models, at last illuminated how ATP1B3 negative regulated NLRP3 inflammasome activation, which may Lay a theoretical foundation for NLRP3 inflammasome activation as new target of sepsis treatment.

炎症小体过度激活导致炎性因子过度释放和免疫细胞焦亡，在脓毒症发生发展中起重要作用，寻找相应的负调控药物并阐明机制具有重要意义。我们前期报道氯喹能够抑制NLRP3炎症小体激活和巨噬细胞焦亡，可提高内毒素血症小鼠存活率，但具体机制不明。预实验发现氯喹抑制NLRP3炎症小体活化关键信号—钾离子外流，转录组结果提示氯喹上调钾离子转运蛋白ATP1B3 mRNA水平。结合文献分析，本项目推测上调钾离子转运蛋白ATP1B3表达负调控LPS介导的巨噬细胞NLRP3炎症小体活化，减少炎症因子释放和巨噬细胞焦亡，发挥对脓毒症的保护作用。为证实该假设，项目拟在细胞模型和脓毒症动物模型中干预ATP1B3研究其通过signal1和signal2通路调控NLRP3炎症小体，进而保护脓毒症动物模型，最终明确ATP1B3负调控NLRP3炎症小体在脓毒症中的作用，为基于NLRP3炎症小体为脓毒症治疗靶点的药物研究奠定基础。

炎症小体过度激活导致炎性因子过度释放和免疫细胞焦亡，在脓毒症发生发展中起重要作用，寻找相应的负调控药物并阐明机制具有重要意义。本项目在前期研究基础上提出本项目推测上调钾离子转运蛋白ATP1B3表达负调控LPS介导的巨噬细胞NLRP3炎症小体活化，减少炎症因子释放和巨噬细胞焦亡，发挥对脓毒症的保护作用。为证实该假设，项目拟在细胞模型和脓毒症动物模型中干预ATP1B3研究其对NLRP3炎症小体活化的调控作用及机制。通过以上研究发现：（1）氯喹和柚皮素抑制NLRP3炎症小体活化Caspase-1、IL-1β和IL-18成熟释放，以及巨噬细胞焦亡。（2）药物抑制Atp1b3转录和表达，负调控NLRP3炎症小体。（3）通过研究过表达或低表达ATP1B3的巨噬细胞模型，明确ATP1B3调控NLRP3炎症小体蛋白复合物组装，抑制NLRP3炎症小体。（4）通过构建低表达ATP1B3的内毒素血症小鼠模型研究，明确低表达ATP1B3促进内毒素血症小鼠炎症和损伤。（5）DPI通过抑制E.coli介导的巨噬细胞ATP生成，通过ATP-P2X7R信号通路抑制NLRP3炎症小体，减轻器官功能损伤，提高感染模型小鼠生存率。